Martin Gibbs

The regulation of starch metabolism by inorganic phosphate.

Summary The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spinach chloroplast are investigated. During CO 2 fixation in the light increasing levels of Pi (0–1.0 mM), which have little effect on the rate of fixation, prolong the lag of starch synthesis and reduce its maximal rate from 14.5 to 6.8 μg-atom C incorporated into starch per milligram chlorophyll per hour. The label in dihydroxyacetone phosphate and other sugar phosphates, especially sedoheptulose-1,7-bis-phosphate, increases up to 10-fold under these conditions. Starch degradation in the dark is monitored in the chloroplast by following the breakdown of the 14 C labeled starch. Pi concentrations in the order of 2.5–5.0 mM are optimal for starch mobilization at a rate of about 1 μg-atom C per milligram chlorophyll per hour.

Effect of photosynthesis, photosynthetic inhibitors and oxygen on the activity of ribulose 5-phosphate kinase.

Abstract Ribulose 5-P kinase is activated up to 4 fold by intact photosynthesizing spinach chloroplasts or by dark incubation of broken chloroplasts with dithiothreitol. The activation by light is blocked by O 2 , arsenite, iodoacetamide and by dichlorophenyldimethylurea.

H2 metabolism in photosynthetic organisms II. Light-dependent H2 evolution by preparations from Chlamydomonas, Scenedesmus and spinach☆

Abstract Light-dependent H 2 evolution from dithiothreitol as electron donor was observed with cell-free preparations of anaerobically adapted Chlamydomonas reinhardii, Scenedesmus obliquus and from spinach chloroplasts mixed with Chlamydomonas hydrogenase. NADH substituted for dithiothreitol as electron donor only in the Chlarmydomonas preparation. Dibromothymoquinone, an antagonist of plastoquinone, selectively inhibited H 2 photoevolution from NADH. These results are interpreted as indicating that 3-(3,4-dichlorophenyl)-1,1-dimethyl urea insensitive H 2 photoevolution by algae containing hydrogenase is due to the capability of NADH to reduce plastoquinone in the electron transport chain, and to evolve H 2 by a low redox potential carrier of photosystem I.

Partial purification and characterization of two fructose diphosphate aldolases from Chlamydomonas mundana

Abstract 1. 1.|Two fructose-1,6-diphosphate aldolases (EC 4.1.2.7) have been detected in extracts of the unicellular green alga Chlamydomonas mundana . The two activities can be resolved by gel filtration of crude cell extracts on Sephadex G-200. 2. 2.|A metal-requiring enzyme has been purified 30-fold from cells cultured in the light on acetate. The enzyme is markedly stimulated by Fe 2+ and is strongly inhibited by metal-chelating agents. The Michaelis constant for the enzyme is 3 · 10 -4 M fructose diphosphate. 3. 3.|An enzyme not dependent upon a metal for activity has been highly purified from cells cultured with carbon dioxide by elution of the activity from cellulose phosphate columns with fructose diphosphate. The purified enzyme is unaffected by high concentrations of chelating agents and is strongly inhibited by heavy metals. The Michaelis constant is 4 · 10 -5 M fructose diphosphate.

Respiration of sugars in spinach (Spinacia oleracea), maize (Zea mays), and Chlamydomonas reinhardtii F-60 chloroplasts with emphasis on the hexose kinases

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate of CO2 release. The pH optimum for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2 release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO2 release from glucose in the Chlamydomonas chloroplast about 70%. 14CO2 evolution from [1–14C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stromal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity.

Photoregulation of Fructose and Glucose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii F-60 and Spinach

The photoregulation of chloroplastic respiration was studied by monitoring in darkness and in light the release of 14CO2 from whole chloroplasts of Chlamydomonas reinhardtii F-60 and spinach (Spinacia oleracea L.) supplied externally with [14C] glucose and [14C]-fructose, respectively. CO2 release was inhibited more than 90% in both chloroplasts by a light intensity of 4 W m-2. Oxidants, oxaloacetate in Chlamydomonas, nitrite in spinach, and phenazine methosulfate in both chloroplasts, reversed the inhibition. The onset of the photoinhibitory effect on CO2 release was relatively rapid compared to the restoration of CO2 release following illumination. In both darkened chloroplasts, dithiothreitol inhibited release. Of the four enzymes (fructokinase, phosphoglucose isomerase, glucose-6-P dehydrogenase, and gluconate-6-P dehydrogenase) in the pathway catalyzing the release of CO2 from fructose, only glucose-6-P dehydrogenase was deactivated by light and by dithiothreitol.

PHOTOSYNTHESIS IN THE ALGAE

The classical kinetic experiments of Calvin and his associates utilizing 14C0, need not be discussed here. They have been described in detail in the publication of Bassham and Ca1vin.l Their experiments involved chiefly Chlorella and Scenedesmus. Similar tracer patterns were described by Norris and associates 2 for other green algae including Haematococcus, Spirogyra and Chlorococcus; the blue-green algae, Phormidium, Synedchococcus, and Nostoc; and the red alga, Porphyridium. The photosynthetic carbon reduction cycle involves 12 enzymically catalyzed steps. The first complete enzyme profile of this cycle was reported by Peterkofsky and Racker in extracts of photoautotrophically grown Chlorella and Euglena. They found lower amounts of ribulose 1,s-dip carboxylase, transaldolase, sedoheptulose 1,7-diphosphatase, and fructose 1,6-diphosphatase than expected from the overall photosynthetic rate. Latzko and Gibbs extended their study by comparing the rate of photosynthetic CO, fixation with the enzyme activities of the cycle in Euglena during chloroplast development and in Chlorella pyrenoidosa grown photoautotrophically in the presence and absence of glucose or heterotrophically with glucose as the sole source of carbon. The activities of several enzymes in extracts of Euglena and Chlorella were not sufficient to satisfy the observed rate of photosynthesis (TABLES 1 & 2) . These included ribulose 1 ,S-diP carboxylase, fructose 1,6-diphosphatase, sedoheptulose 1,7-diphosphatase, and transaldolase. However, information other than enzyme profile studies lead to the conclusion that at least ribulose 1,s-dip carboxylase and fructose 1,6-diphosphatase are involved in CO, fixation. For example, the activities of the carboxylase and the diphosphatase paralleled changes in CO, fixation caused by

Inhibition of CO2 fixation in intact spinach chloroplasts by 3-phosphoglyceric acid☆

Glycerate-3-P inhibits CO2 fixation of isolated spinach chloroplats at concentrations higher than 1 mM but does not inhibit O2 evolution. Glycerate-3-P inhibition of photosynthesis is not overcome by higher bicarbonate concentrations.

Water Deficit Effects on Non-Stomatal Mediated Photosynthesis

In several recently published reviews dealing with water deficit effects on photosynthesis (Farquhar, Sharkey, 1982; Hanson, Hitz, 1982) the possibility was raised that stress induced lesions other than increased stomatal resistance to CO2 uptake might at least partially contribute to inhibitions of photosynthesis in water stressed crop plants. Supporting this contention, Plaut (1971) has found that reduced reaction medium ψπ (simulating in vivo cellular dehydration) inhibited photosynthesis in isolated chloroplasts. These data indicate that dehydration adversely affects the basic biochemical processes which facilitate CO2 assimilation.

Inhibition of non-reversible d-glyceraldehyde-3-phosphate dehydrogenase from pea shoots by l-glyceraldehyde-3-phosphate

Abstract A non-reversible d -glyceraldehyde-3-phosphate dehydrogenase which does not require phosphate for activity has been partially purified from pea shoots. The substrates for the enzyme are d -glyceraldehyde-3-phosphate and NADP. Strong inhibition is caused by l -glyceraldehyde-3-phosphate.